Clones of mouse fibroblasts that contain hybrid DNA molecules composed of sequences from polyoma virus DNA, the prokaryotic plasmid pBR322 and the yeast leu2 gene will be examined. These cells should survive in leucine-free medium because the recombinant DNA contributes genetic information for an enzyme involved in leucine biosynthesis. This functional gene provides the basis for the biological selection of cells containing recombinant DNA. The studies proposed here will define the potential of this polyoma-leu2-pBR322 hybrid DNA as a eukaryotic cloning vehicle. The number of copies per cell, and whether recombinant DNA has integrated into the host cell DNA or replicates autonomously, will be established. A physical map of restriction enzyme sites on one isolate will be constructed and used to identify sites at which additional sequences may be inserted into future experiments. The number of cells producing polyoma early (T) antigen will be determined by indirect immunofluorescence. The cold labile yeast enzyme beta-isopropylmalate (beta-IPM) dehydrogenase can be identified and quantitated in cell extracts by photometrically assaying the production of alpha-ketoisocaproic acid from beta-IPM. Experiments will be conducted to determine optimum conditions for growth of recombinant DNA-containing cells, and for transfection of mouse fibroblasts and other eukaryotic cells.